Pathogenic Bacillus anthracis in the progressive gene losses and gains in adaptive evolution

Background: Sequence mutations represent a driving force of adaptive evolution in bacterial pathogens. It is especially evident in reductive genome evolution where bacteria underwent lifestyles shifting from a free-living to a strictly intracellular or host-depending life. It resulted in loss of function mutations and/or the acquisition of virulence gene clusters. Bacillus anthracis shares a common soil bacterial ancestor with its closely related bacillus species but is the only obligate, causative agent of inhalation anthrax within the genus Bacillus. The anthrax-causing Bacillus anthracis experienced the similar lifestyle changes. We thus hypothesized that the bacterial pathogen would follow a compatible evolution path. Results: In this study, a cluster-based evolution scheme was devised to analyze genes that are gained by or lost from B. anthracis. The study detected gene losses/gains at two separate evolutionary stages. The stage I is when B. anthracis and its sister species within the Bacillus cereus group diverged from other species in genus Bacillus. The stage II is when B. anthracis differentiated from its two closest relatives: B. cereus and B. thuringiensis. Many genes gained at these stages are homologues of known pathogenic factors such those for internalin, B. anthracis-specific toxins and large groups of surface proteins and lipoproteins. Conclusion: The analysis presented here allowed us to portray a progressive evolutionary process during the lifestyle shift of B. anthracis, thus providing new insights into how B. anthracis had evolved and bore a promise of finding drug and vaccine targets for this strategically important pathogen

Production of a conserved adhesin by the human gastroduodenal pathogen Helicobacter pylori.

An adhesin protein with an approximate subunit molecular weight of 19,600 has been purified from the gastric pathogen Helicobacter pylori. The protein was loosely associated with the cell surface and was removed by gentle stirring or shearing. Released aggregates of the 19.6-kDa protein were removed from suspension by ultracentrifugation and separated from contaminating membranes by washing in 1.0% sodium dodecyl sulfate (SDS). The SDS-insoluble protein was purified further by Mono Q anion-exchange column chromatography. Electron microscopy of the purified adhesin demonstrated that it formed amorphous aggregates similar to the material attached to the bacterial cells and that the aggregates were morphologically distinct from typical fimbriae. Western blot (immunoblot) analysis with antiserum raised against the purified protein from one strain reacted with a protein with a similar subunit molecular weight present in all nine strains of H. pylori examined, but the protein was not present in other Helicobacter species examined. The N-terminal sequences of the 19.6-kDa protein purified from three different strains of H. pylori were identical for the first 28 amino acids, with the 10 amino-terminal residues showing limited sequence homology with the TcpA pilus protein of Vibrio cholerae. The H. pylori 19.6-kDa protein associated both with human and rabbit erythrocytes and with human buccal epithelial cells. Polystyrene microspheres coated with the protein agglutinated human, horse, and rabbit erythrocytes, suggesting that this protein species could mediate adhesion between H. pylori and eucaryotic cells. This ability to act as an adhesin may make this protein an important virulence factor for H. pylori and hence a potential target for a vaccine and therapy

Isolation and biochemical and molecular analyses of a species-specific protein antigen from the gastric pathogen Helicobacter pylori.

A protein of Mr 26,000 which was present in large quantities in extracts of cells of Helicobacter pylori was purified to homogeneity by ammonium sulfate precipitation followed by gel filtration and reversed-phase chromatography or anion-exchange chromatography. The protein appeared to be associated with the soluble fraction of the cell, and antibodies raised against the protein were reactive with whole-cell lysates of a variety of H. pylori strains in a simple immunodot blot assay. This reaction was species specific. Protein sequence determination of the amino terminus and internal cyanogen bromide fragments and amino acid composition analysis were performed. An oligonucleotide derived from these data was used to clone a fragment encoding most of the coding sequence. Expression in Escherichia coli was dependent on vector promoters. The DNA sequence of the fragment was determined. DNA probes derived from the cloned fragment hybridized to genomic DNA of all H. pylori strains tested, but not to DNAs of Helicobacter mustelae, Wolinella succinogenes, various Campylobacter species, and a panel of gram-negative enteric bacteria. The apparent uniqueness of this protein may be exploited for the development of species-specific diagnostics for this gastric pathogen

Antigenic diversity of the S-layer proteins from pathogenic strains of Aeromonas hydrophila and Aeromonas veronii biotype sobria.

The antigenic relatedness of paracrystalline surface array proteins with subunit molecular weights of approximately 52,000 from isolates of Aeromonas hydrophila and Aeromonas veronii biotype sobria belonging to a single heat-stable serogroup was examined. Enzyme-linked immunosorbent assay and immunoblotting with two different polyclonal antisera against surface exposed and non-surface-exposed epitopes of the S-layer protein from A. hydrophila TF7 showed that the S-layer proteins of the mesophilic aeromonads were antigenically diverse. NH2-terminal amino acid sequence analysis of four antigenically different proteins showed that while the proteins were structurally related, they differed in primary sequence. Absorption experiments with heterologous live cells showed that cross-reactive epitopes were in non-surface-exposed regions of the S-layer proteins, while absorption with homologous live cells showed that the immunodominant epitopes of the S-layer protein of strain TF7 were strain specific and exposed on the surface of the native, tetragonal array produced by this strain. Proteolytic digestion of the TF7 S-layer protein with trypsin, chymotrypsin, or endoproteinase Glu-C produced an amino-terminal peptide of approximate Mr 38,000 which was refractile to further proteolytic cleavage under nondenaturing conditions. This peptide carried the immunodominant surface-exposed region of the protein, and chemical cleavage with cyanogen bromide further mapped the portion of these surface-exposed epitopes to a peptide of approximate Mr 26,000, part of which maps within the Mr 38,000 protease-resistant NH2-terminal peptide